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Eukaryotic Cell, July 2007, p. 1239-1247, Vol. 6, No. 7
1535-9778/07/$08.00+0     doi:10.1128/EC.00414-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of a Highly Efficient Gene Targeting System Induced by Transient Repression of YKU80 Expression in Candida glabrata ^,

Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan,¹ Suzuka National College of Technology, Shiroko, Suzuka, Mie 510-0294, Japan²

Received 30 December 2006/ Accepted 4 May 2007

In the pathogenic yeast Candida glabrata, gene targeting to generate knockouts and "knockins" is a potentially powerful method for the analysis of gene function. Its importance increased after the C. glabrata genome sequence project, but progress in the field is hampered by inefficient mechanisms for gene targeting. With the use of 40-bp homologous flanking DNA, no gene targeting was identified. To address this issue, YKU80 was disrupted, leading to an increase in targeting efficiency of 5.1% using 40-bp flanking homologous DNA. To harness the beneficial effects of YKU80 inactivation on gene targeting frequency without incurring any negative effects, such as synthetic sickness or lethality, we developed a new system whereby the expression of YKU80 was restored following a transient knockdown of expression during transformation. Strains used for this new system carried a SAT1 flipper in the YKU80 promoter region, which was used to repress expression during transformation but was spontaneously excised from the locus after the transformation. By using this strain, DNA damage induced by methyl methane sulfonate, H₂O₂, UV irradiation, and hydroxyurea before and during gene targeting was evaluated and the mutation rate of URA3 was determined. No significant effects of the SAT1 flipper on these processes have been identified. After the SAT1 flipper is excised, a 34-bp FLP recombination target sequence is left in the promoter region. However, the levels of mRNA transcription were restored and no difference in the survival ratio in vivo compared to that with the YKU80 wild-type strain was identified.

Published ahead of print on 18 May 2007.

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